Role Of Second Messengers Research Articles

Knockout of the PKC Substrate Pleckstrin Causes Pleomorphic Defects in Platelets, Lymphocytes and Granulocytes.

Pleckstrin is a 40 kDa phosphoprotein containing amino- and carboxyl-terminal Pleckstrin Homology (PH) domains separated by a DEP domain. Pleckstrin's expression is restricted to platelets and leukocytes, and represents approximately 1% of total cellular protein within these cells. Following platelet and leukocyte activation, PKC rapidly phosphorylates pleckstrin inducing it to bind membrane bound phospholipids such as phosphatidylinositol 4,5 bisphosphate (PIP2). Heterologously expressed phosphorylated pleckstrin colocalized with integrins and induces cytoskeletal reorganization. To better define the role of pleckstrin in vivo, we introduced a loss-of-function mutation into the murine pleckstrin gene. Pleckstrin-null mice were present in offspring at a frequency consistent with a Mendelian inheritance pattern. Adult pleckstrin −/− mice had 32% lower platelet counts than their littermates, but exhibited no spontaneous hemorrhage. Given the role of PKC and phospholipid second messengers on cytoskeletal dynamics, and our observations of pleckstrin overexpression in cell lines, we analyzed whether loss of pleckstrin affected cell spreading. Pleckstrin −/− platelets spread extremely poorly upon immobilized fibrinogen, and rarely exhibited broad membrane extensions. Granulocytes from pleckstrin −/− mice also have a spreading defect, as well as impaired ability to generate reactive oxygen species in the response to TNFα. Knockout B-cells, CD4-T-cells, and CD8-T-cells all migrated approximately 30% as efficiently as wild type cells in response to a gradient of SDF-1α in a transwell assay. These data suggest that loss of pleckstrin causes cytoskeletal defects in cells of multiple hematopoietic lineages. Analyzing whether this caused a functional defect, we found that pleckstrin −/− platelets exhibited a 22% dense- and 24% alpha-granule exocytosis defect, and a 35% defect in thrombin-induced calcium entry. In spite of these abnormalities, platelets changed shape and aggregated normally after stimulation with thrombin, ADP, or collagen in vitro. Pleckstrin knockout platelets did have a markedly impaired aggregation response following exposure to the PKC stimulant, PMA. This suggested that pleckstrin is a critical effector for PKC-mediated aggregation, but another pathway is able to compensate for this loss of pleckstrin following agonist stimulation. We reasoned that the alternative pathway might also utilize PIP2-dependent second messengers. Since the phosphorylation of PIP2 by PI3K generates second messengers that also contribute to platelet aggregation, we tested whether PI3K compensated for the loss of pleckstrin. We found that the PI3K inhibitor, LY294002 profoundly impaired the aggregation of pleckstrin knockout platelets in response to stimulation of the thrombin receptor. In contrast, the PI3K inhibitor minimally affected wild type platelets. This demonstrates that second messengers generated by PI3K are able to compensate for loss of pleckstrin. This also demonstrates that thrombin-induced platelet aggregation can be mediated by one of two parallel pathways, one involving PKC and pleckstrin, and the other involving PI3K. Together, our results show that pleckstrin is an essential component of PKC-mediated platelet activation and signals directed to the cytoskeleton.

Effects of the perfusion flow rate on skeletal muscle energy metabolism and a possible role of second messengers in this process

The metabolic turnover in the isolated in vitro perfused and superfused rat skeletal muscle (musculus gracilis cranialis) was enhanced by increasing the medium flow rate under relaxed conditions. In a recent study we have measured the tissue concentrations of second messengers: cyclic adenosine 3'5'-monophosphate (cAMP), cyclic guanosine 3'5'-monophosphate (cGMP), and D-myo-inositol 1,4,5-trisphosphate (IP3) under similar experimental conditions to analyze their potential role in the described stimulation of metabolic rate by changes of perfusion flow rate. The tissue levels of the two second messengers' cAMP and cGMP were not significantly changed after increasing the perfusion flow rate and they probably have no transduction role in the induced alteration of skeletal muscle metabolism. However, the IP3 content was extremely reduced after increasing flow rate. This decrease in the tissue concentration of IP3 induced by increasing the flow rate indicates the possible role of IP3 in this signal transduction, leading to changes in the cellular metabolic pathways.

Hépatite C, Interféron α et dépression : principales hypothèses physiopathologiques

Hépatite C, Interféron α et dépression : principales hypothèses physiopathologiques

Genistein reduces agonist-induced contractions of porcine coronary arterial smooth muscle in a cyclic AMP-dependent manner

Low concentrations of genistein enhance the vasodilatation induced by endothelium-independent vasodilators. The present study examined whether or not low concentrations of genistein modulate contractions in isolated porcine coronary arteries. The role of second messengers in the response to genistein was also assessed. Arterial rings were studied in organ baths and contracted with KCl, U-46619 (9,11-dideoxy-9α, 11α-methanoepoxy prostaglandin F 2α), 5-hydroxytryptamine (5-HT) or endothelin-1 in the absence or presence of genistein (≤3 μM). Genistein significantly reduced agonist-induced but not KCl-induced contraction. Inhibition of endothelial nitric oxide synthase and disruption of endothelial function by Triton-X100 did not affect the modulation of contraction by genistein. The genistein-induced attenuation of contraction could be mimicked by both cAMP and cGMP analogs. However, only the cAMP-dependent protein kinase inhibitor, Rp-8-Br-cAMPS, abolished the effect of genistein. These results suggest that genistein reduces agonist-induced contraction by an endothelium-independent manner. This action is mediated via the cAMP-dependent signal transduction pathway.

Assaying phosphoinositide phosphatases.

The roles of phosphoinositide second messengers as signaling molecules in a vast array of cellular processes including cell growth, metabolism, vesicular transport, programmed cell death, and responses to extracellular signals are only beginning to be understood. The recent identification of novel phosphoinositide signaling molecules underscores the need for methodology with which to characterize the enzymes responsible for regulating cellular phosphoinositide levels. One of the ways in which cells control these lipids is through dephosphorylation by phosphoinositide phosphatases, which oppose and regulate the actions of phosphoinositide kinases. We describe herein two rapid and simple assays for characterizing phosphoinositide phosphatases that can be used to provide a basis for understanding the activity and specificity of these enzymes.

Cyclic ADP-ribose is a second messenger in the lipopolysaccharide-stimulated proliferation of human peripheral blood mononuclear cells.

Cyclic ADP-ribose (cADPR), a universal calcium mobilizer from intracellular stores, was recently demonstrated to stimulate proliferation of various cell types. The role of cADPR in a specific process of monocyte- and plasma-mediated activation of T-lymphocytes by lipopolysaccharide (LPS) was addressed using human mononuclear cells from peripheral blood (PBMCs). Incubation of PBMCs with 0.1 microg/ml of LPS for 24 h provided a doubling in the intracellular levels of cADPR as compared with unstimulated PBMCs. The cADPR increase was abolished either by prior removal of monocytes or by pre-incubating a whole PBMC population with a monoclonal antibody against the monocyte marker CD14. The increased concentrations of intracellular cADPR elicited by LPS stimulation were paralleled by significant increases in NAD+ levels and in the activities of ectocellular and membrane-bound fractions of ADP-ribosyl cyclase/cADPR hydrolase activities. A cytosolic ADP-ribosyl cyclase was also detectable in PBMCs and its activity was comparably enhanced by LPS stimulation. This soluble cyclase is distinguished from the membrane-bound cyclase by both substrate and inhibitor sensitivities. LPS-stimulated PBMCs showed 2-3-fold increases of intracellular calcium ([Ca2+]i), and these changes were prevented completely by the cADPR antagonist 8-Br-cADPR and by ryanodine. Both compounds, and the cyclase inhibitor nicotinamide, significantly inhibited the T-lymphocyte proliferation induced by LPS in PBMCs. These results demonstrate that cADPR plays a role of second messenger in the adaptive immune recognition process of LPS-stimulated proliferation of PBMCs.

Future perspectives for glycolipid research in medicine.

Medical interest in glycolipids has been mainly directed to the rare and complex glycosphingolipid storage disorders that are principally caused by unitary deficiencies of lysosomal acid hydrolases. However, glycolipids are critical components of cell membranes and occur within newly described membrane domains known as lipid rafts. Glycolipids are components of important antigen systems and membrane receptors; they participate in intracellular signalling mechanisms and may be presented to the immune system in the context of the novel CD1 molecules present on T lymphocytes. A knowledge of their mechanism of action in the control of cell growth and survival as well as developmental pathways is likely to shed light on the pathogenesis of the glycosphingolipid storage disorders as well as the role of lipid second messengers in controlling cell mobility and in the mobilization of intracellular calcium stores (a biological role widely postulated particularly for the lysosphingolipid metabolite sphingosine 1-phosphate). Other sphingolipid metabolites such as ceramide 1-phosphate may be involved in apoptotic responses and in phagocytosis and synaptic vesicle formation. The extraordinary pharmaceutical success of enzymatic complementation for Gaucher's disease using macrophage-targeted human glucocerebrosidase has focused further commercial interest in other glycolipid storage diseases: the cost of targeted enzyme therapy and its failure to restore lysosomal enzymatic deficiencies in the brain has also stimulated interest in the concept of substrate reduction therapy using diffusible inhibitory molecules. Successful clinical trials of the iminosugar N-butyldeoxynojirimycin in type 1 Gaucher's disease prove the principle of substrate reduction therapy and have attracted attention to this therapeutic method. They will also foster important further experiments into the use of glycolipid synthesis inhibitors for the severe neuronopathic glycosphingolipidoses, for which no definitive treatment is otherwise available. Future glycolipid research in medicine will be directed to experiments that shed light on the role of sphingolipids in signalling pathways, and in the comprehensive characterization and their secretory products in relation to the molecular pathogenesis of the storage disorders; experiments of use to improve the efficiency of complementing enzymatic delivery to the lysosomal compartment of storage cells are also needed. Further systematic screening for inhibitory compounds with specific actions in the pathways of glycosphingolipid biosynthesis will undoubtedly lead to clinical trials in the neuronopathic storage disorders and to wider applications in the fields of immunity and cancer biology.

Sexual dimorphism for protein kinase cε signaling in a rat model of vincristine-induced painful peripheral neuropathy

Painful peripheral neuropathy is a major dose-limiting adverse effect of many cancer chemotherapeutic agents, such as the vinca alkaloids and taxanes. Recent studies demonstrate sexual dimorphism in second-messenger signaling for primary afferent nociceptor sensitization, and a role of second messengers in the models of metabolic and toxic painful peripheral neuropathies. This study tested the hypothesis that sexual dimorphism alters the severity and second-messenger signaling pathways for enhanced nociception in an animal model of vincristine-induced painful peripheral neuropathy. I.V. injection of vincristine induced mechanical hyperalgesia that was greater in female rats. Gonadectomy in the females but not the males abolished the sex-dependent difference in mechanical hyperalgesia; this effect of gonadectomy in females was reversed by estrogen replacement. Inhibition of protein kinase Cε (PKCε) attenuated vincristine-induced hyperalgesia in males and ovariectomized females, but not in normal females or in estrogen-replaced ovariectomized females. Inhibitors of protein kinase A, protein kinase G, p42/p44-mitogen activated protein kinase and nitric oxide synthase also attenuated vincristine-induced hyperalgesia, but to a similar degree in both sexes. These data demonstrate an estrogen-dependent sexual dimorphism in vincristine-induced hyperalgesia (female>male) and an unexpected opposite sexual dimorphism in the contribution of PKCε to the severity of this hyperalgesia (male>female).

The roles of calcium/calmodulin-dependent and Ras/mitogen-activated protein kinases in the development of psychostimulant-induced behavioral sensitization.

Although the development of behavioral sensitization to psychostimulants such as cocaine and amphetamine is confined mainly to one nucleus in the brain, the ventral tegmental area (VTA), this process is nonetheless complex, involving a complicated interplay between neurotransmitters, neuropeptides and trophic factors. In the present review we present the hypothesis that calcium-stimulated second messengers, including the calcium/calmodulin-dependent protein kinases and the Ras/mitogen-activated protein kinases, represent the major biochemical pathways whereby converging extracellular signals are integrated and amplified, resulting in the biochemical and molecular changes in dopaminergic neurons in the VTA that represent the critical neuronal correlates of the development of behavioral sensitization to psychostimulants. Moreover, given the important role of calcium-stimulated second messengers in the expression of behavioral sensitization, these signal transduction systems may represent the biochemical substrate through which the transient neurochemical changes associated with the development of behavioral sensitization are translated into the persistent neurochemical, biochemical and molecular alterations in neuronal function that underlie the long-term expression of psychostimulant-induced behavioral sensitization.

Nuclear localisation of cytosolic phospholipase A2-alpha in the EA.hy.926 human endothelial cell line is proliferation dependent and modulated by phosphorylation.

Cytosolic phospholipase A(2)-alpha (cPLA(2)-alpha) is a calcium-sensitive enzyme involved in receptor-mediated eicosanoid production. In resting cells, cPLA(2)-alpha is present in the cytosol and nucleus and translocates to membranes via its calcium-dependent lipid-binding (CaLB) domain following stimulation. cPLA(2)-alpha is also regulated by phosphorylation on several residues, which results in enhanced arachidonic acid release. Little is known about the factors controlling the nuclear localisation of cPLA(2)-alpha. Here the nuclear localisation of cPLA(2)-alpha in the EA.hy.926 human endothelial cell line was investigated. Nuclear localisation was dependent on proliferation, with subconfluent cells containing higher levels of nuclear cPLA(2)-alpha than contact-inhibited confluent or serum-starved cells. The broad-range protein kinase inhibitor staurosporine caused a decrease in the nuclear level of cPLA(2)-alpha, whereas the protein phosphatase inhibitor okadaic acid increased the level of nuclear cPLA(2)-alpha. Using inhibitors for specific mitogen-activated protein (MAP) kinases, both p42/44(MAPK) and p38(MAPK) were shown to be important in modulating nuclear localisation. Finally, inhibition of nuclear import and export using Agaricus bisporus lectin and leptomycin B, respectively, demonstrated that cPLA(2)-alpha contains functional nuclear localisation and export signals. Thus we have identified a novel mode of regulation of cPLA(2)-alpha. This, together with the increasing body of evidence supporting the role of nuclear lipid second messengers in gene expression and proliferation, may have important implications for controlling the growth of endothelial cells in angiogenesis and tumour progression.

Influence of VIP on prolactinemia in turkey anterior pituitary cells: role of cAMP second messenger in VIP-induced prolactin gene expression.

Vasoactive intestinal peptide (VIP) is the avian prolactin (PRL)-releasing factor. In the turkey, hypothalamic VIP immunoreactivity and mRNA content, as well as VIP levels in hypophyseal portal blood, are closely related to the state of prolactinemia and the reproductive stage. The present study investigated the role of VIP on prolactinemia in turkey anterior pituitary (AP) cells through PRL gene expression and the role of a cAMP second messenger system on VIP-induced PRL expression. In primary AP cells harvested from hens in different prolactinemic states, steady state promoter activities were positively correlated with secreted PRL levels. VIP increased PRL promoter activities in AP cells from hens with intermediate PRL levels (laying), but not in AP cells from hypoprolactinemic hens (nonphotostimulated reproductively quiescent). However, in AP cells from hyperprolactinemic hens (incubating), PRL promoter activity was down-regulated by VIP. PRL mRNA steady state levels were significantly decreased by the cAMP analogue, 8-bromo-cyclic adenosine monophosphate (8-Br-cAMP), and PRL secretion was down-regulated by the phosphodiesterase blocker, 3-isobutyl-1-methylxanthine (IBMX) in a dose-dependent manner, suggesting that the cAMP second messenger system might be involved in the inhibitory action of dopamine upon VIP-stimulated PRL secretion and gene expression at the pituitary level. In a study of VIP immediate and long-term effects on c-fos expression in relation to PRL expression, VIP dramatically induced c-fos mRNA expression within 5 min, suggesting that VIP-induced c-fos expression might be involved in VIP-stimulated PRL secretion and gene expression. These results provide additional evidence of the functional significance of VIP in PRL gene expression and suggest that changes in PRL promoter activity by VIP may be one of the important inductive mechanisms leading to prolactinemia.

Pharmacological regulation of the late steps of exocytosis.

We used amperometry to analyze the role of several second messengers and drugs in the exocytotic kinetics of bovine chromaffin cells. Activation of PKG produces a slowing down of exocytosis, which is not generally accompanied by changes in the net granule content of catecholamines. These effects are also observed after mild PKA activation. However, strong PKA stimulation also causes an increase in the apparent granule content of catecholamines, suggesting the presence of composed fusion. Conversely, PKC activation promotes acceleration of the exocytotic process. We also analyzed the contribution of different Ca(2+) channel subtypes to the exocytotic kinetics at the single event level. Although N-subtype channels do not contribute to total catecholamine release, their blockade produces a slowing down of exocytosis without changes in granule content. However, L or P/Q blockade causes, in addition, a reduction in the apparent granule content. The L-type agonist BAY-K-8644 produces giant secretory amperometric spikes, indicating that Ca(2+) favors composed fusion prior to exocytosis. Our data suggest that second messengers continuously regulate exocytotic kinetics and granule content. In addition, several well-known antihypertensive agents, such as sodium nitroprusside, organic nitrates, hydralazine, or Ca(2+) antagonists, could be acting through these novel mechanisms on sympathetic synapses by changing the synaptic performance, thereby producing additional vasodilatory effects.

Characterization of calcium oscillations in normal and benzo[a]pyrene-treated clone 9 cells.

Intracellular Ca2+ oscillations induced by oxytocin and vasopressin were analyzed in a rat liver cell line (Clone 9) in order to identify mechanisms by which benzo[a]pyrene (BaP) alters Ca2+ signaling patterns in these cells. Clone 9 cells exhibit an initial Ca2+ spike, followed by Ca2+ oscillations upon oxytocin or vasopressin treatment. The range of frequencies (maximum 110 mHz) was dependent on agonist concentration with a constant amplitude less than or equal to the amount of Ca2+ generated from the inositol trisphosphate (InsP(3))-sensitive pool. This study examined contributions of extracellular and intracellular pools to the frequency of Ca2+ oscillations and the role of membrane channels, second messengers, and different pharmacological reagents on the regulation of oscillation frequency in both control and BaP-treated cells. Results indicated that the Ca2+ oscillations are mainly due to inositol 1,4,5-triphosphate (InsP(3))-sensitive stores and that extracellular Ca2+ contributes to refilling of this intracellular Ca2+ pool. The frequency of Ca2+ oscillations is also sharply affected by protein kinase C activated by phospholipase C. In BaP-treated Clone 9 cells, basal Ca2+ levels were elevated and the frequency of Ca2+ oscillations was suppressed in a dose-dependent fashion. Suppression of Ca2+ oscillations is due, at least in part, to an effect of BaP on enhanced opening of K+ channels. This was confirmed by showing that inhibition of the K+ channel opening by tetraethylammonium chloride can reverse the effect of BaP on oxytocin-induced Ca2+ oscillations, and potentially decrease the toxicity of BaP.

Myo-Inositol 1,4,5-trisphosphate and Ca(2+)/calmodulin-dependent factors mediate transduction of compression-induced signals in bovine articular chondrocytes.

Although the effects of mechanical loading on chondrocyte metabolic activities have been extensively characterized, the sequence of events through which extracellular mechanical signals are transduced into chondrocytes and ultimately modulate cell activities is not well understood. Here, studies were performed to map out the sequential intracellular signalling pathways through which compression-induced signals modulate aggrecan mRNA levels in bovine articular chondrocytes. Bovine articular cartilage explants were subjected to a compressive stress of 0.1 MPa for 1 h in the presence or absence of inhibitors or antagonists of the phosphoinositol and Ca(2+)/calmodulin signalling pathways in order to determine the roles of second messengers and effector molecules of these pathways in transducing the compression-induced signals. In the absence of the inhibitors, aggrecan mRNA levels were stimulated by compression 2-4-fold relative to levels in tare-loaded (see below) explants. Treatment of the explants with graded levels of the protein kinase C inhibitor chelerythrine or bisindolylmaleimide I, followed by 1 h compressive loading, did not significantly alter the load-induced elevation of aggrecan mRNA levels. In contrast, thapsigargin, which depletes the Ins(1,4,5)P3-sensitive intracellular Ca(2+) stores, completely blocked the load response without significantly altering aggrecan mRNA levels in tare-loaded explants. Similarly, antagonists of the Ca(2+)/calmodulin signalling pathway dose-dependently or completely blocked the load-response. The results obtained demonstrate that transduction of the compression-induced aggrecan mRNA-regulating signals requires Ins(1,4,5)P3- and Ca(2+)/calmodulin-dependent signalling processes in bovine articular chondrocytes.

Myo-Inositol 1,4,5-trisphosphate and Ca2+/calmodulin-dependent factors mediate transduction of compression-induced signals in bovine articular chondrocytes

Although the effects of mechanical loading on chondrocyte metabolic activities have been extensively characterized, the sequence of events through which extracellular mechanical signals are transduced into chondrocytes and ultimately modulate cell activities is not well understood. Here, studies were performed to map out the sequential intracellular signalling pathways through which compression-induced signals modulate aggrecan mRNA levels in bovine articular chondrocytes. Bovine articular cartilage explants were subjected to a compressive stress of 0.1MPa for 1h in the presence or absence of inhibitors or antagonists of the phosphoinositol and Ca2+/calmodulin signalling pathways in order to determine the roles of second messengers and effector molecules of these pathways in transducing the compression-induced signals. In the absence of the inhibitors, aggrecan mRNA levels were stimulated by compression 2–4-fold relative to levels in tare-loaded (see below) explants. Treatment of the explants with graded levels of the protein kinase C inhibitor chelerythrine or bisindolylmaleimide I, followed by 1h compressive loading, did not significantly alter the load-induced elevation of aggrecan mRNA levels. In contrast, thapsigargin, which depletes the Ins(1,4,5)P3-sensitive intracellular Ca2+ stores, completely blocked the load response without significantly altering aggrecan mRNA levels in tare-loaded explants. Similarly, antagonists of the Ca2+/calmodulin signalling pathway dose-dependently or completely blocked the load-response. The results obtained demonstrate that transduction of the compression-induced aggrecan mRNA-regulating signals requires Ins(1,4,5)P3- and Ca2+/calmodulin-dependent signalling processes in bovine articular chondrocytes.

Design and synthesis of peptide antagonists and inverse agonists for G protein-coupled receptors

Publisher Summary This chapter describes the approaches to the design and synthesis of antagonists in detail and discusses the way by which the same principles can be used to synthesize an inverse agonist. While it is theoretically possible to design a drug that binds perfectly to the receptor, it may be difficult to, at same time, design a molecule with perfect biodistribution, diffusion, pharmacokinetics, and others. In any case, G protein-coupled receptor (GPCR) antagonist ligands will continue to play a major role in the evaluation of these receptors and as drugs. As science begins to understand the role of second messengers in the body, antagonists will be used to help balance the effects of the second messengers on some receptors to correct dysfunctions of mind and body. When ligands interact with the extracellular portion of the receptor, a binding process occurs and there is a conformational change in the receptor, which, in the case of the agonist, causes the intracellular portion to interact with G proteins. G protein components then trigger various second messengers leading to signal transduction.

Interaction between serotonin and nitric oxide (NO) in the activation of the serotoninergic system in the common snail.

The effects of serotonin and NO donors on serotoninergic neurons (more than 60) in the brain of the common snail Helix lucorum were studied. Serotonin and NO donors induced depolarization in all neurons. and increased spike activity and activated the synchronous synaptic input, including train-like input. resulting in the onset of synchronous train activity in all these neurons. The excitatory effect of serotonin was significantly decreased by 5,7-dihydroxytryptamine (5.7-DOT) and monomethylarginine--a blocker of endogenous NO synthesis. Both these substances blocked the serotonin activation of the synchronous train input. 5.7-DOT also blocked the activation of this input by NO donors. but had no effect on their excitatory actions. The effects of 5,7-DOT developed quickly, were reversible, and were comparable to the effects of serotonin receptor antagonists. The data obtained here provide evidence that serotonin and NO have similar regulatory effects on the serotoninergic system in the snail brain. Not only do they excite serotoninergic neurons, but they also coordinate their functioning by activating common synaptic inputs. which are apparently also serotoninergic. It is suggested that NO has the role of a second messenger during serotoninergic excitation and functions as a co-transmitter for the presynaptic input.

Production of Phosphatidylinositol 3,4,5-Trisphosphate and Phosphatidic Acid in Platelet Rafts: Evidence for a Critical Role of Cholesterol-Enriched Domains in Human Platelet Activation

Glycosphingolipid- and cholesterol-enriched membrane microdomains, called rafts, can be isolated from several mammalian cells, including platelets. These microdomains appear to play a critical role in signal transduction in several hematopoietic cells, but their function in blood platelets remains unknown. Herein, we first characterized the lipid composition, including the fatty acid composition of phospholipids, of human platelet rafts. Then their role in platelet activation process was investigated. Interestingly, thrombin stimulation led to morphological changes of rafts correlating with the production of lipid second messengers in these microdomains. Indeed, we could demonstrate for the first time that a large part of the stimulation-dependent production of phosphatidic acid and phosphoinositide 3-kinase products was concentrated in rafts. Moreover, cholesterol depletion with methyl-beta-cyclodextrin disrupted platelet rafts, dramatically decreased the agonist-dependent production of these lipid signaling molecules, and impaired platelet secretion and aggregation. Cholesterol repletion restored the physiological platelet responses. Altogether our data indicate that rafts are highly dynamic platelet membrane structures involved in critical signaling mechanisms linked to the production of lipid second messengers. The demonstration of phosphatidylinositol 3,4,5-trisphosphate production in rafts may have general implications for the understanding of the role of this key second messenger found ubiquitously in higher eucaryotic cells.

Autocrine role of adrenomedullin in the human adrenal cortex.

Previous studies from our laboratory have reported that adrenomedullin is synthesised in rat zona glomerulosa cells. In the present studies, it was found that the human adrenocortical cell line H295R expresses the gene encoding adrenomedullin, and that immunoreactive adrenomedullin is released into the culture medium. Furthermore, it was found that secretion of adrenomedullin is regulated by angiotensin II and forskolin. Studies on the actions of adrenomedullin and calcitonin gene-related peptide (CGRP) revealed a stimulatory effect of adrenomedullin, but not of CGRP, on aldosterone and cortisol secretion. These data suggest that adrenomedullin is not acting by a CGRP receptor-mediated mechanism in the H295R cell line. Adrenomedullin was also found to increase cAMP production, suggesting that in the adrenal, as in other cell types, cAMP is a second messenger for adrenomedullin action. However, the effects of adrenomedullin were not fully mimicked by forskolin, possibly suggesting a role for an additional second messenger. The presence of mRNA encoding both the putative adrenomedullin receptors, L1 and calcitonin receptorlike receptor/receptor-associated modulatory protein 2 (CRLR/RAMP-2), was demonstrated in H295R cells, but RAMP-1 was not detected, suggesting that these cells do not express the CGRPI receptor CRLR/RAMP-1. Taken together, these data have demonstrated that adrenomedullin is synthesised and secreted by H295R cells. The observed rate of adrenomedullin synthesis suggests that this peptide exerts a paracrine/autocrine effect in this adrenocortical cell line, probably acting through a specific adrenomedullin receptor, to stimulate steroidogenesis and increase aldosterone synthase expression.

N-acetylneuraminic acid (NANA) stimulates in situ cyclic AMP production in tentacles of sea anemone (Aiptasia pallida): possible role in chemosensitization of nematocyst discharge.

Cnidocytes, the stinging cells of cnidarians, optimally discharge nematocysts in response to combined physical contact and stimulation of specific chemoreceptors. In the tentacles of certain sea anemones, the primary chemoreceptors bind N-acetylated sugars, such as N-acetylneuraminic acid (NANA). Sensitization with NANA predisposes contact-sensitive mechanoreceptors (CSMs) to trigger discharge in response to physical contact. In the ectoderm of sea anemone tentacles, cnidocyte/supporting cell complexes (CSCCs) control and trigger nematocyst discharge. Previous findings have implicated cyclic AMP (cAMP) as a second messenger in NANA-sensitized nematocyst discharge. However, no reports have directly demonstrated that the cAMP content of tentacles changes in response to NANA stimulation. We now show that NANA elevates in situ cAMP levels in a dose-dependent manner in the ectoderm of tentacles from the sea anemone Aiptasia pallida. However, the endoderm of tentacles shows no detectable cAMP response to NANA. The effect of NANA on the cAMP content of the ectoderm is biphasic. Micromolar NANA increases the in situ cAMP level, with a maximal response occurring at 1.8x10(-5)mol x l(-1) NANA. At higher NANA concentrations, the cAMP content decreases to that of controls. Because the cAMP dose/response curve to NANA coincides precisely with the dose/response curves of NANA-sensitized nematocyst discharge and nematocyst-mediated adhesive force, a second-messenger role for cAMP in NANA-sensitized nematocyst discharge is strongly suggested. The addition of isobutyl-1-methylxanthine (IBMX) to the medium with sea anemones increases tissue cAMP levels both in the absence and in the presence of NANA. However, anesthetizing anemones in sea water containing high levels of Mg(2+) blocks the NANA-stimulated cAMP response of the ectoderm. In addition, our results suggest that NANA-stimulated cAMP may activate endogenous cAMP-dependent protein kinase (PKA) in broken cell preparations of tentacles. Thus, NANA-stimulated cAMP may function as a second messenger in the NANA chemosensory signaling pathway controlling nematocyst discharge.